THE REGULATION OF HEPATIC CHOLINE TRANSPORT by Rebecca Yaworski The University of Ottawa A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies In Biochemistry November 2017 © Rebecca Yaworski, Ottawa, Canada, 2017 TABLE OF CONTENTS 1 ACKNOWLEDGEMENTS .......................................................................................................... 2 LIST OF FIGURES ........................................................................................................................ i v 3 LIST OF ACRONYMS ................................................................................................................ ii v 4 ABSTRACT .................................................................................................................................... v v 5 INTRODUCTION .......................................................................................................................... 1 iii 5.1 The History of Choline ......................................................................................... 1 5.2 Choline Transport ................................................................................................. 3 5.3 Choline Transporter Like Protein family ............................................................. 5 5.3.1 CTL1 ............................................................................................................. 5 5.3.2 CTL2-5 .......................................................................................................... 6 5.4 CTL1 and Choline Metabolic Pathways .............................................................. 8 5.5 Phosphatidylcholine and the Liver ..................................................................... 12 5.6 Choline and Metabolic Disease .......................................................................... 13 5.7 The Circadian Rhythm and Liver Metabolism ................................................... 15 5.7.1 The Circadian Rhythm ................................................................................ 16 5.7.2 Circadian disruption and liver metabolism ................................................. 17 5.8 Rationale and Objectives .................................................................................... 21 6 EXPERIMENTAL PROCEDURES ....................................................................................... 22 6.1 Animals .............................................................................................................. 22 6.2 Tissue Harvest and RNA Isolation ..................................................................... 22 6.3 cDNA synthesis .................................................................................................. 23 6.4 Real-time qPCR.................................................................................................. 24 6.5 Murine Primary Hepatocyte Isolation ................................................................ 25 6.6 Rate of Hepatic Choline Uptake......................................................................... 27 6.7 CTL1-5 Uptake Inhibition Assay ....................................................................... 28 6.8 Saturation Kinetics of the CTL1-5 Choline Transporter System ....................... 29 6.9 Compound Preparation and Treatment of Primary Hepatocytes ....................... 30 6.10 Circadian Time Trials ..................................................................................... 31 6.11 Bligh & Dyer Lipid Extraction ....................................................................... 31 6.12 Blood Serum Choline Quantification Assay .................................................. 32 6.13 Tissue based Chromatin Immunoprecipitation ............................................... 33 6.13.1 Cross-linking and Chromatin Preparation .................................................. 33 ii 6.13.2 Chromatin Preclearing and Immunoprecipitation ....................................... 34 6.13.3 Washing and Elution ................................................................................... 35 6.13.4 DNA Purification ........................................................................................ 36 6.14 BCA Protein Quantification Assay ................................................................. 36 6.15 Western blots .................................................................................................. 37 6.16 In Vivo Choline Uptake .................................................................................. 39 6.17 Adeno Virus Treatment of Primary Hepatocytes ........................................... 39 6.18 Procedure for the Removal of Obese and Time Dependent Mouse Livers .... 40 6.19 Bioinformatics specification ........................................................................... 41 6.20 Statistical Analyses ......................................................................................... 41 7 RESULTS ....................................................................................................................................... 42 7.1 Kinetics of Choline Transporters in Primary Hepatocytes................................. 42 7.2 A Comparison of Choline Transporters and Metabolizing Enzymes between Primary Hepatocytes and Liver Homogenate ............................................................... 44 7.3 Treatment Effect on Hepatic Choline Uptake and Incorporation into Phosphatidycholine ....................................................................................................... 45 7.4 Effects of Oleate Treatment on Hepatic Choline Metabolism ........................... 47 7.5 Obesity Affects CTL1 mRNA Expression and Choline Incorporation.............. 48 7.6 CTL1 Expression and Liver Choline Uptake are Influenced by the Circadian Rhythm. ......................................................................................................................... 50 7.7 CHKα and CTL1 mRNA Expression show 24 hour Rhythmicity. .................... 51 7.8 Bioinformatics analysis indicates 4 potential RORE consensus sequences for REV-ERBα/RORα binding upstream of the SLC44A1 gene ....................................... 53 7.9 Disrupting the Circadian Rhythm Affects Liver Choline Incorporation and SLC44A1 Expression.................................................................................................... 56 7.10 Both Obesity and the Circadian Rhythm Affect Choline Uptake and CTL1 Expression. .................................................................................................................... 57 7.11 The Effect of Obesity and Circadian Rhythm on the Transcription of Choline Metabolizing Enzymes and the OCT1 Transporter. ..................................................... 58 8 DISCUSSION ............................................................................................................................... 60 8.1 CTL1-5 is an intermediate choline transport system responsible for 80% of hepatic uptake. .............................................................................................................. 60 8.2 Choline incorporation is increased following treatment with oleate.................. 62 8.3 Oleate stimulates PC synthesis in hepatocytes but does not affect CTL1 expression. .................................................................................................................... 64 8.4 Obesity Decreases CTL1 mRNA Expression and Choline Incorporation ......... 66 8.5 CTL1 expression and function are regulated by the circadian rhythm. ............. 68 iii 8.6 Bioinformatics indicates that the CTL1 promoter contains REV-ERBα/RORα binding sites. ................................................................................................................. 69 8.7 BMAL knockout affects both CTL1 expression and hepatic choline uptake. ... 71 8.8 Obesity decreases the overall uptake and incorporation of choline in the morning. ........................................................................................................................ 72 8.9 Obesity suppresses CHKα and PEMT expression in the morning. .................... 73 9 CONCLUDING REMARKS .................................................................................................... 74 10 REFERENCES ............................................................................................................................. 76 11 APPENDIX 1 ................................................................................................................................ 86 11.1 Breeding Scheme Towards the Generation of a CTL1-/- Mouse ..................... 86 11.2 Preliminary Study Evaluating the Effect of Obesity on Hepatic CTL1 Expression. .................................................................................................................... 90 iv ACKNOWLEDGEMENTS Morgan I am truly grateful for your guidance and patience over the past couple years, I have not only advanced my abilities as a scientist but have gained a greater understanding of what it means to be a scholar and professional. Thank you for always leaving your door open, encouraging me to ask questions and also making me find the answers on my own. I wish you and your family all the best in the future. To my friends in the lab and in the office, thank you for all the laughs, good times and interesting conversations!